Glycoengineering of the methylotrophic yeast Hansenula polymorpha for the production of glycoproteins with trimannosyl core N ‐glycan by blocking core oligosaccharide assembly

The initial lipid‐linked oligosaccharide Glc 3 Man 9 GlcNAc 2 ‐dolichyl pyrophosphate (Dol‐PP) for N ‐glycan is synthesized and assembled at the membrane of the endoplasmic reticulum (ER) and subsequently transferred to a nascent polypeptide by the oligosaccharide transferase complex. We have identified an ALG3 homolog ( HpALG3 ) coding for a dolichyl‐phosphate‐mannose dependent α‐1,3‐mannosyltransferase in the methylotrophic yeast Hansenula polymorpha. The detailed analysis of glycan structure by linkage‐specific mannosidase digestion showed that HpALG3 is responsible for the conversion of Man5GlcNAc 2 ‐Dol‐PP to Man 6 GlcNAc 2 ‐Dol‐PP, the first step to attach a mannose to the lipid‐linked oligosaccharide in the ER. The N ‐glycosylation pathway of H. polymorpha has been remodeled by deleting the HpALG3 gene in the Hpoch1 null mutant strain blocked in the yeast‐specific outer mannose chain synthesis and by introducing an ER‐targeted Aspergillus saitoi α ‐1,2‐mannosidase gene. This glycoengineered H. polymorpha strain produced glycoproteins mainly containing trimannosyl core N ‐glycan (Man 3 GlcNAc 2 ), which is the common core backbone of various human‐type N ‐glycans. The results demonstrate the high potential of H. polymorpha to be developed as an efficient expression system for the production of glycoproteins with humanized glycans.